Protein engineering
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Protein engineering. Tailor-made biocatalysts The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity

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Protein engineering
Protein engineering

Tailor-made biocatalysts

  • The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity

  • Rational (re)design versus directed evolution


Protein engineering1
Protein engineering

Genetic manipulation techniques

  • Large-scale supply of enzymes at reasonable price

  • Identification of new biocatalysts (screening) doesnot always yield suitable enzymes for a given synthetic problem

  • Computer-aided site-directed mutagenesis

  • Directed (molecular) evolution


Protein engineering2
Protein engineering

Site-directed mutagenesis

  • Requires structural information and knowledge about relationship between sequence, structure, function and mechanism

  • Very information-intensive

  • Rapid progress in NMR / X-ray methods

  • Genome sequence information

  • Molecular modeling, bioinformatics

  • Prediction of selectivity, activity, stability etc.


Protein engineering3
Protein engineering

Rational redesign strategy

  • Protein structure

  • Planning of mutants, SDM

  • Vectors containing mutated genes

  • Transformation in E. coli

  • Protein expression and purification

  • Mutant enzyme analysis

  • Negative mutants

  • Improved mutant enzymes


Protein engineering4
Protein engineering

Rational redesign

  • Amino acid substitutions often selected by sequence comparison with homologous sequences

  • Results have to be carefully interpreted

  • Minor changes by a single point mutation may cause significant structural disturbance

  • Comparison of 3D-structure of mutant and wild-type enzyme necessary


Protein engineering5
Protein engineering

Inversion of stereospecificity of VAO

  • Current Opinion in Chemical Biology (2001)

  • A very nice study on alteration of enantioselectivity based on structural comparison of two members of structurally related FAD-dependent oxidoreductases, of which one is (R)-specific and the other (S)-specific

  • Site-directed mutagenesis introduced (S)-selectivity in the (R)-selective wild-type enzyme

  • Structural analysis of the mutant enzyme revealed that the mutations are really site-directed


Protein engineering6
Protein engineering

Directed evolution

  • Evolutive biotechnology, molecular evolution

  • Random mutagenesis of the gene encoding the biocatalyst (e.g. by error-prone PCR)

  • DNA shuffling: recombination of gene fragments (staggered extension process or random priming recombination)


Protein engineering7
Protein engineering

Directed evolution strategy

  • Random mutagenesis

  • Library of mutated genes

  • Transformation in E. coli

  • Mutant library > 10.000 clones

  • Protein expression in microtiter plates

  • Selection parameters

  • Mutant enzyme and product analysis

  • In vitro-recombination, transformation etc.


Protein engineering8
Protein engineering

Selection parameters

  • Substrate range

  • Stability in organic solvent

  • Stability towards reaction conditions

  • Thermal stability

  • High-throughput product analysis

  • Robot technology


Protein engineering9
Protein engineering

Selection parameters

  • Hydrolysis of esters: agar-plate assay based on pH indicators

  • Parallel assaying of replica-plated colonieswith substrate analog

  • Isotopically labeled substrates

  • Capillary electrophoresis (7000 samples per day)

  • Optimization with saturation mutagenesis


Protein engineering10
Protein engineering

Digital image screening

  • Naphthalene hydroxylation by P450cam

  • Co-expression of horseradish peroxidase

  • Fluorescent products amenable by digital screening

  • P450 hydroxylation of indole to indigo

  • Inversion of enantioselectivity

  • Increase of peroxidase specificity with guaiacol


Protein engineering11
Protein engineering

Improving thermostability

  • Cold-adapted proteases

  • Combined screening for activity, thermostability, organic solvent tolerance and pH-profile

  • Engineering of entire metabolic pathways

  • Phytoene desaturase and lycopene cyclase shuffling for carotenoid biosynthesis

  • Molecular breeding


Protein engineering12
Protein engineering

Biochemistry

Vanillyl-alcohol oxidase

  • Production of natural vanillin

    2-Hydroxybiphenyl monooxygenase

  • Large-scale production of substituted catechols

    Galactose oxidase

  • Production of new oligosaccharides


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